Scientific Abstract: We lack a basic understanding how spontaneous autoimmune disease arises. Studies of antigen-tolerized transgenic NOD mice have suggested the primacy of different autoantigens in initiating the autoimmunity that leads to type 1 diabetes (T1D). However, theoretical considerations based on our knowledge of T cell tolerance induction predict that beta-cell autoimmunity should be initially lost to many beta-cell antigens simultaneously but there is little experimental evidence in support of this scenario. We have developed a modified ELISPOT assay that enables the characterization of autoreactive T cells within the pancreatic lymph nodes (PLN) and islet-infiltrating T cell population. Our preliminary data indicate that antigen-specificity and phenotype of autoreactive T cells within the target tissue is quite different from the current picture that was obtained through analysis of T cell populations in the periphery of NOD mice. We expect that our further longitudinal characterization of autoreactive CD4+ and CD8+ T cell responses in the PLN, islets and spleen will lead to a new conceptualization of the autoimmune processes in NOD mice. Our analysis of the functional avidity of early T cell autoreactivity in the PLN and islets will provide an independent line of evidence to support either the "hierarchal" or the "simultaneous" model of the development of T cell autoimmunity. The lessons learned will support/refute the hypothesis that autoimmunity can be circumvented by tolerizing individuals to "initiating" target antigens. Our studies will also elucidate the relationship between peripheral T cell responses and those in the target tissue. As only PBMC are available from humans, understanding the relationship between T cell autoreactivities in the periphery and target tissue is important to assess the feasibility of using T cell-based markers to monitor disease progression, the efficacy of interventive therapies, and the status of transplanted islets. Laypersons: We lack a basic understanding how spontaneous autoimmune disease arises. We have established the ability to directly characterize autoreactive T cells within the pancreatic lymph nodes (PLN, wherein autoimmunity is thought to first arise) and the islet-infiltrating T cell population, which heretofore has not been feasible. We expect that the results of this proposal will lead to a new conceptualization of the autoimmune process in NOD mice. The same basic processes may also occur in human organ-specific autoimmune diseases. The results will help guide our thinking on potential therapeutic approaches, as well as the utility of biomarkers based on peripheral T cell responses. [unreadable] [unreadable] [unreadable]